Soluble TNFR1 Research Articles

Superoxide anion-induced pain and inflammation depends on TNFα/TNFR1 signaling in mice

Inhibition of tumor necrosis factor-alpha (TNFα) and superoxide anion production reduces inflammation and pain. The present study investigated whether superoxide anion-induced pain depends on TNFα signaling and the role of superoxide anion in TNFα-induced hyperalgesia to clarify the interrelation between these two mediators in the context of pain. Intraplantar injection of a superoxide anion donor (potassium superoxide) induced mechanical hyperalgesia (0.5–5h after injection), neutrophil recruitment (myeloperoxidase activity), and overt pain-like behaviors (paw flinching, paw licking, and abdominal writhings) in wild-type mice. Tumor necrosis factor receptor 1 deficiency (TNFR1-/-) and treatment of wild-type mice with etanercept (a soluble TNFR2 receptor that inhibits TNFα actions) inhibited superoxide anion-induced pain-like behaviors. TNFR1−/− mice were also protected from superoxide anion donor-induced oxidative stress, suggesting the role of this pathway in the maintenance of oxidative stress. Finally, we demonstrated that Apocynin (an NADPH oxidase inhibitor) or Tempol (a superoxide dismutase mimetic) treatment inhibited TNFα-induced paw mechanical hyperalgesia and neutrophil recruitment (myeloperoxidase activity). These results demonstrate that TNFα/TNFR1 signaling is important in superoxide anion-triggered pain and that TNFα/TNFR1 signaling amplifies the oxidative stress triggered by superoxide anion, which contributes to sustaining pain and inflammation.

The Flavonoid Wogonin Reduces CLL Cell Survival in Vitro and Leukemia Development in Eµ-TCL1 Mice By Targeting Aberrant TNF Receptor Signaling

The importance of the tumor microenvironment in the development of B-cell chronic lymphocytic lekuemia (CLL) is now widely accepted. Previous studies within our and other groups revealed the establishment of an inflammatory milieu in CLL characterized by enhanced expression and secretion of several cytokines and their receptors. Using 250 CLL serum samples and 50 age-matched controls, we found significantly increased levels of the TNFα receptors TNFR1 and TNFR2 in the patients that positively correlated with an adverse clinical outcome. Based on these findings we aimed to investigate the functional role of TNFR signaling in CLL development.In contrast to healthy B cells that do not express TNFα receptors, we detected TNFR1 expression in CLL cells upon survival-inducing culture conditions and in lymph node sections of 80 CLL patients, where TNFR1 signals co-localized to the B cell marker CD20 and were mainly present within proliferation centers. These findings were confirmed in Eµ-TCL1 mice, a well-established mouse model for CLL. Here, CLL cells in the peripheral blood were negative for TNFR1. However, the cells upregulated the receptor upon entering the spleen where they get into contact with accessory cells, receive survival stimuli and undergo proliferation. In addition, increased levels of soluble TNFR1 in the serum were confirmed in the mice.After ligand binding, TNFR1 can activate two different signaling pathways: (1) the extrinsic apoptosis cascade through its death receptor domain, or (2) survival-inducing NFκB signaling. The latter pathway can be blocked by wogonin, a naturally occurring monoflavonoid. In a multitude of in vitro and in vivo studies, wogonin has been shown to exert anti-oxidant, anti-inflammatory and anti-tumor activities. By ex vivo treatment of CLL cells with TNFα we observed NFκB activation which was reversed by TNFα-blocking antibody, suggesting TNFR1-mediated survival signaling in CLL. Therefore, we aimed to test whether wogonin can prevent this signaling in CLL cells and might therefore represent a novel potential drug for CLL.In CLL cocultures, wogonin treatment resulted in a concentration-dependent apoptosis induction, which was significantly increased by the addition of TNFα. To test the effects of wogonin in vivo, we transplanted isogenic, immunocompetent wild-type mice with CLL cells from leukemic TCL1 animals and treated them daily with 40 mg/kg wogonin. When treatment was started 2 days after transplantation, wogonin significantly reduced spleen weights and lead to a reduced CLL content in the spleen, the bone marrow and the peritoneal cavity. If treatment was started in advanced disease stage, wogonin slightly lowered spleen weight and the CLL content in the spleen, whereas the percentage of CLL cells in the peripheral blood was increased. Interestingly, here wogonin treatment resulted in a loss of cell surface TNFR1 expression in splenic CLL cells and increased TNFR1 levels in the serum. These data suggest that wogonin induces a redistribution of CLL cells in vivo, preventing their homing to lymphoid organs and that loss of TNFR1 expression might be involved in this process.In summary, our results show that TNFR1 signaling is involved in CLL cell activation and survival. Targeting this pathway with wogonin reduces CLL cell viability in vitro and leukemia development in TCL1 mice. DisclosuresNo relevant conflicts of interest to declare.

S141 Tumour Necrosis Factor Receptor 1 Shedding Is Related To Acute Skeletal Muscle Wasting In Critical Illness

Introduction Muscle wasting occurs early and rapidly in critically ill patients. It results from a decrease in muscle protein synthesis and an increase in its breakdown, with muscle necrosis a common associated finding. The drivers for such atrophy and necrosis remain poorly understood, as do the related regulatory pathways. We hypothesised that systemic and intracellular cytokines play a role in this process. Methods The UK-MUSCLE study prospectively studied the wasting response (change in Rectus Femoris cross sectional area (RF CSA ) using serial ultrasound) in critically ill patients admitted to the ICU. Cytokine profiles (high sensitivity cytokine chip array, Randox, Ireland) were analysed in serum samples from 62 of these patients (days 1 and 7) and in contemporaneous Vastus Lateralis biopsies of 35 patients. Tumour Necrosis Factor (TNF)-α, TNF receptor (TNFR) 1 and 2, interleukin (IL)1a, il1b, IL-2, IL-4, IL-6, IL-8, IL-10, Vascular Endothelial growth Factor (VEGF), Interferon (IFN)-γ, Monocyte Chemoattractant Protein-1(MCP-1) and Epidermal Growth Factor were assayed. Muscle necrosis was determined by hematoxylin and eosin staining of the Vastus Lateralis biopsies. Results Intramuscular TNFR1 concentrations increased over 7 days (0.83 ± 1.1 µg/L to 2.07 ± 2.65 µg/L; p = 0.042), as did intramuscular interleukin-10 (22.69 ± 26.5 ng/L to 59.8 ± 80.0 ng/L; p = 0.005). Increases in serum IL-1a (0.52 ± 0.26 ng/L to 0.57 ± 0.28 ng/L, p = 0.03), VEGF (166.86 ± 231.7 ng/L to 246.6 ± 236.7 ng/L, p CSA was related to that in intramuscular cytokines by linear and logistical-regression analysis, using 10% loss as a cut off. Loss in RF CSA over 10 days was very weakly correlated with serum TNFR1 concentration on days 1 (r 2 =0.12; p 2 =0.09; p = 0.02) and with serum IL-10 concentration (r 2 =0.19; p Discussion Soluble TNFR1 is associated with the degree of muscle wasting in critical illness. This relationship may be causal as TNF1R signalling leads to activation of nuclear factor kappa beta and apoptosis. Whilst no evidence was seen for intramuscular inflammation, increased intramuscular IL-10 may be protective, in its anti-inflammatory role.

Expression of CXCR1 (interleukin-8 receptor) in murine macrophages after staphylococcus aureus infection and its possible implication on intracellular survival correlating with cytokines and bacterial anti-oxidant enzymes.

Interaction with the live Staphylococcus aureus promotes secretion of interleukin-8 (IL-8), although the expressions of functional CXCR1 (IL-8RA) in murine macrophages have not been identified. Expression of CXCR1 was induced in S. aureus-infected macrophages, whereas, CXCR1 was undetectable in control macrophages. CXCR1 blocking significantly reduced the phagocytosis of S. aureus and TNF-α, IL-6, IL-1β, IFN-γ, IL-12, and IL-8 production and increased release of MIP-2 and soluble TNF-R1. Increased bacterial catalase and decreased superoxide dismutase (SOD) activities by S. aureus with concomitant decrease in hydrogen peroxide (H2O2), superoxide anion, and nitric oxide (NO) release were observed in case of prior CXCR1 blocking. In the presence of cytochalasin D, S. aureus-mediated induction of IL-8 was inhibited concomitant with decreased bacterial count suggesting that internalization of S. aureus was necessary for induction of IL-8. Shedding of TNF-R1 due to CXCR1 blocking after S. aureus inoculation was critical for neutralization of TNF-α signaling and arrests the inflammation.

Pro-inflammatory cytokines and soluble receptors in response to acute psychosocial stress: Differential reactivity in bipolar disorder

Mounting evidence suggests a chronic pro-inflammatory state in individuals with bipolar disorder (BD). Stress exposure is known to exacerbate several inflammatory conditions as well as psychiatric disorders. Here, we analyzed plasma levels of pro-inflammatory cytokines and their soluble receptors to realistic acute psychosocial stress challenge in BD. Thirteen euthymic type 1 BD patients and 15 matched controls underwent the Trier Social Stress Test protocol (TSST). Blood samples were collected before and after TSST and plasma cytokines interleukin IL-2, IL-6, IL-33, and tumor necrosis factor alpha (TNF-α) were measured. In addition TNF-α soluble receptors TNFR1 and TNFR2, and IL-33 soluble receptor sST2 were assessed. Increased IL-33 and reduced sST2 levels were observed in BD subjects as compared to controls, independently of stress exposure. Following TSST, there were higher levels of IL-2 and reduced levels of sTNFR1 in both groups. However, the magnitude change for both cytokines was found higher in controls than BD subjects. Our data suggest that BD patients have differential stress reactivity as compared to controls, possibly related to an immunologic imbalance and failure of regulatory mechanisms.

AB0268 Association between Soluble Interferon Receptor and Various Biomarkers in Patients with Rheumatoid Arthritis

BackgroundLarge numbers of inflammatory cytokines are considered to be complicatedly involved in rheumatoid arthritis (RA), which may be affected by unbalanced homeostasis of natural killer cells or Th1 and Th2...

Low level tumor necrosis factor-alpha protects cardiomyocytes against high level tumor necrosis factor-alpha: brief insight into a beneficial paradox.

Whether tumor necrosis factor-alpha (TNFα) caused beneficial or detrimental cardiovascular effects remains poorly defined. Anti-TNFα agents improved cardiac end points in chronic rheumatic diseases characterized by progressive deterioration of cardiac function. In contrast, anti-TNFα agents did not always improve but actually worsened cardiac function in non-rheumatic patients with heart failure (HF), in spite of that HF usually accompanies with high circulating levels of TNFα. To shed light on these mixed findings, we characterized the effects of TNFα in H9c2 cardiomyocytes. Cells were incubated for 24h with increasing concentrations of TNFα, hydrogen peroxide, aminotriazole, or etoposide. Posttreatment cell viability was assessed by antimycin A-inhibitable reduction of 3-(4,dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and the IC50 value of each test compound was defined. H9c2 cells were also preconditioned with a low non-toxic concentration of TNFα and then re-challenged with increasing concentrations of TNFα and other stressor agents. In re-challenge experiments, all of the IC50 values increased significantly, with the IC50 value of TNFα increasing approximately 16-fold. TNFα preconditioning increased cardiomyocytes shedding of the external portion of transmembrane type 1 and type 2 TNFα receptors [(soluble TNFα receptors (sTNFR)]. Levels of survival-oriented soluble TNFR2 (sTNFR2) always exceeded those of death-oriented sTNFR1. When exposed to TNFα at its IC50 value, preconditioned cardiomyocytes showed an increased release of sTNFR2 but not sTNFR1. These results denoted that preconditioning by "low TNFα" helped cardiomyocyte to withstand toxicity from "high TNFα" or other agents. These results also suggested that beneficial or detrimental effects of anti-TNFα agents might well depend on whether these agents spared or intercepted discrete amounts of TNFα that preconditioned cardiomyocytes and made them more resistant to high concentrations of TNFα.

Hypothalamo–pituitary–adrenal axis, glucose metabolism and TNF-α in narcolepsy

Narcolepsy with cataplexy is caused by a deficiency in the production of hypocretin, which regulates sleep and wakefulness and also influences appetite, neuroendocrine functions and metabolism. In this case-control study 11 patients with narcolepsy and cataplexy and 11 healthy adults underwent an oral glucose tolerance test and dexamethason-inhibition cortisol releasing hormone stimulation test. The average age of patients and controls was 35.1 ± 13.2 and 41.0 ± 2.9 years, respectively, the body mass index (BMI) 28.1 ± 6.6 and 25.5 ± 4.7. We did not find evidence of a significantly increased prevalence of disturbed glucose tolerance in narcolepsy patients. After hypothalamo-pituitary–adrenal axis suppression, the number of non-suppressors did not differ between the groups, indicating normal negative feedback sensitivity. The level of cortisol after dexamethason suppression was significantly lower in narcoleptic patients suggesting a slight basal down-regulation and/or a slightly increased negative feedback sensitivity of the major endocrine stress system in narcolepsy. Following CRH stimulation, there were no significant differences in levels of ACTH or cortisol, and in adrenocortical responsivity to ACTH. Finally, narcoleptic patients displayed significantly higher plasma levels of TNF- α , solubleTNFR p55, soluble TNFR p75 and IL-6 after adjustment for BMI. The present study confirms that narcolepsy by itself is not associated with disturbances of glucose metabolism, but goes along with a subtle dysregulation of inflammatory cytokine production. We also found that dynamic HPA system response is not altered, whereas negative feedback to dexamethasone might be slightly enhanced. This study was supported by the grant of the Czech Ministry of Health NT 13238-4/2012, PRVOUK-P26/LF1/4 and by the European Union Framework 6 (MCRTN-CT-2004-512362).

TNFR1/TNF-α and mitochondria interrelated signaling pathway mediates quinocetone-induced apoptosis in HepG2 cells

Quinocetone, a new quinoxaline 1, 4-dioxide derivative, has been widely used as an animal feed additive in China. This study was conducted to explore the molecular mechanisms of apoptosis induced by quinocetone in HepG2 cells. MTT assay revealed that the viability of HepG2 cells was significantly inhibited by quinocetone in a dose- and time-dependent manner. Quinocetone-induced apoptosis in HepG2 cells was characterized by cell and nuclei morphology change, cell membrane phosphatidylserine translocation, DNA fragmentation, cleavage of poly (ADP-ribose) polymerase (PARP) and a cascade activation of caspase-8, caspase-9 and caspase-3. Simultaneously, quinocetone induced HepG2 cell cycle arrest, which was supported by overexpression of p21. Cytochrome c release was caused by the mitochondrial membrane potential dissipation, a process related to quinocetone-induced Bid cleavage and elevated Bax/Bcl-2 ratio. Moreover, quinocetone treatment caused the up-regulation of TNF-α and TNFR1 in HepG2 cells. Both soluble TNFR1 receptors and caspase inhibitors suppressed quinocetone-induced apoptosis. In addition, the protein levels of p53, p-p38 and p-JNK were increased in quinocetone-treated cells. Taken together, quinocetone induced apoptosis in HepG2 cells via activation of caspase, interaction of TNF-α and TNFR1 and modulation of the protein levels of Bid, Bax and Bcl-2, involving the participation of p53, p38 and JNK.

Human Skin Mast Cells Express Complement Factors C3 and C5

We examine whether complement factor C3 or C5 is synthesized by human skin-derived mast cells and whether their synthesis is regulated by cytokines. C3 and C5 mRNAs were assessed by RT-PCR, and proteins by flow cytometry, confocal microscopy, Western blotting, and ELISA. C3 and C5 mRNAs were each expressed, and baseline protein levels/10(6) cultured mast cells were 0.9 and 0.8 ng, respectively, and located in the cytoplasm outside of secretory granules. C3 accumulated in mast cell culture medium over time and by 3 d reached a concentration of 9.4 ± 8.0 ng/ml, whereas C5 levels were not detectable (<0.15 ng/ml). Three-day incubations of mast cells with IL-1α, IL-1β, IL-17, IFN-γ, IL-6, or anti-FcεRI did not affect C3 protein levels in culture medium, whereas incubations with PMA, TNF-α, IL-13, or IL-4 enhanced levels of C3 1.7- to 3.3-fold. In contrast with C3, levels of C5 remained undetectable. Importantly, treatment with TNF-α together with either IL-4 or IL-13 synergistically enhanced C3 (but not C5) production in culture medium by 9.8- or 7.1-fold, respectively. This synergy was blocked by attenuating the TNF-α pathway with neutralizing anti-TNF-α Ab, soluble TNFR, or an inhibitor of NF-κB, or by attenuating the IL-4/13 pathway with Jak family or Erk antagonists. Inhibitors of PI3K, Jnk, and p38 MAPK did not affect this synergy. Thus, human mast cells can produce and secrete C3, whereas β-tryptase can act on C3 to generate C3a and C3b, raising the likelihood that mast cells engage complement to modulate immunity and inflammation in vivo.

Novel Biomarkers for the Progression of Diabetic Nephropathy: Soluble TNF Receptors

Despite 2 decades of advances in therapy of diabetic patients, the prevalence of diabetic nephropathy among patients with diabetes has not decreased. However, large-scale multicenter studies have achieved great success in terms of the reduction of albuminuria, suggesting that albuminuria might not be an accurate surrogate marker for slowing the rate of renal function decline. It is important to be able to identify individuals at high risk for renal function decline, or ultimately, end-stage kidney disease (ESKD) and its associated cardiovascular disease (CVD). More sensitive early biomarkers, other than albuminuria and the estimated glomerular filtration rate (eGFR), should be required. Recently, serum concentrations of soluble tumor necrosis factor (TNF), receptor 1 (TNFR1), and TNFR2 have predicted future GFR loss and ESKD in patients of a wide variety of stages and both types of diabetes. Longitudinal interventional studies are needed to validate these biomarkers in a broad range of populations prior to implementation in routine diabetes management.

Mutations in the TNFRSF1A, CEBPE and ELANE genes in a Congenital Cyclic Neutropenia Patient: A New Syndrome?

Abstract 11Cyclic neutropenia (CyN) is a hematologic disorder in which blood cell counts particularly granulocytic neutrophil numbers show cycles at 21 day intervals. The majority of CyN patients (ca 90 %) harbor inherited mutations in the ELANE gene. Intriguingly, same ELANE mutations are present in two different hematologic syndromes: congenital as well as in cyclic neutropenias. It is unclear how mutation in the same gene cause congenital or cyclic neutropenia. We aimed to identify genes which are exclusively mutated in cyclic or in congenital neutropenia additionaly to the ELANE gene mutations. Recently, we found in congenital neutropenia patients additional to ELANE mutations inherited mutations in for example the G6PC3 gene or the HAX1 gene (Germeshausen, M., et al, Haematologica 2010). This suggests cooperating effects of different defective intracellular signaling pathways and excludes that mutated ELANE alone is responsible for the pathogenesis of congenital or cyclic neutropenia.To identify gene mutations causing cyclic neutropenia in association with ELANE mutation, we performed whole genome sequencing using Complete Genomics technology (Complete Genomics. Inc, Mountain View, CA.) of a family with an affected CyN patient. The CyN patient harbors sporadic heterozygous ELANE mutation (c.761C>G p.W241L) and her family consists of a healthy brother and healthy parents. We identified a novel heterozygous point mutation in the tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A) gene (c.664C>T; p.R121Q) in the affected patient and her mother. This mutation was confirmed by Sanger sequencing. The TNFRSF1A gene encodes p55 subunit of the TNFa receptor (TNFR1) and intriguingly this gene is known to be frequently mutated in patients with Tumour necrosis factor receptor-associated periodic fever syndrome (TRAPS), a disease clinically similar to cyclic neutropenia. TRAPS is an autosomal dominant disorder characterized by episodes of fever, inflammation and periodical changes in the neutrophil counts. Functional studies in patients with TRAPS described heterogenous effects of different TNFRSF1A mutations on the surface expression and PMA-induced clevage of TNFR1 on the neutrophilic granulocytes and monocytes. In our CyN patient we measured elevated mRNA levels of TNFR1 on neutrophils, in comparison to her healthy family members and unrelated healthy individuals. However, we detected diminished surface expression of the TNFR1 protein, but elevated PMA-induced receptor shedding in the affected CyN patient, in comparison to healthy individuals, as assessed by estimation of soluble TNFR1 in supernatants of PMA-stimulated neutrophils.We also identified in the same patient and her father a second novel heterozygous point mutation in the CEBPE gene (c.636C>A; p.L155M), which was also confirmed by Sanger sequencing. This C>A substitution changes CTG to ATG creating a new start site for translation of a novel isoform of C/EBPε protein. C/EBPε is a myeloid-specific transcription factor playing an important role in granulopoiesis. Mutations in the CEBPE gene have been described in patients with neutrophil-specific granule deficiency (SGD).In summary, we identified additional to the ELANE mutation two novel mutations in a CyN patient, one in the TNFRSF1A gene inherited from the mother, another in the CEBPE gene inherited from the father. Mutations in both genes are already described in patients with TRAPS (periodic fever syndrome) and granulocyte abnormalities, respectively. These mutations in association with the ELANE gene mutation may contribute to the pathogenesis of cyclic neutropenia in this patient. Whether the combination of these three mutations might be responsible for a subgroup of CyN patients remains to be investigated. Disclosures:No relevant conflicts of interest to declare.

Stromal Fibroblast–Specific Expression of ADAM-9 Modulates Proliferation and Apoptosis in Melanoma Cells In Vitro and In Vivo

ADAMs are members of the zinc metalloproteinase superfamily characterized by the presence of disintegrin and metalloprotease domains. In human melanoma, ADAM-9 is expressed in focalized areas of the tumor-stroma border in both melanoma and stromal cells. However, the role of ADAM-9 in melanoma progression remains elusive. To analyze the role of stromal-derived ADAM-9 for the growth and survival of melanoma cells, we have used in vitro coculture systems of melanoma cells and ADAM-9(-/-) fibroblasts. Coculture of melanoma cells in the presence of ADAM-9(-/-) fibroblasts led to increased melanoma cell proliferation and reduced apoptosis as compared with control cocultures. We identified TIMP-1 and sTNFRI as the two relevant factors expressed in increased amounts in culture supernatants from ADAM-9(-/-) fibroblasts. TIMP-1 was associated with induced melanoma cell proliferation, whereas soluble TNFR1 mediated the reduced cellular apoptosis in vitro. In vivo, injection of murine melanoma cells into the flank of ADAM-9(-/-) animals resulted in the development of significantly larger tumors than in wild-type animals as a result of increased proliferation and decreased apoptosis of melanoma cells. Taken together, stromal expression of ADAM-9 during melanoma development modulates the expression of TIMP-1 and sTNFR1, which in turn affect tumor cell proliferation and apoptosis.

P018 Membrane-bound TNF induces protective cell-mediated immune responses to M. bovis BCG infection: Regulation of transmembrane TNF and TNF receptors mediating protection

TNF is a pleiotropic cytokine associated with the development of human immunopathologies and involved in host defense mechanisms. TNF blockade has confirmed the crucial and non-redundant role of TNF in the control and resistance to tuberculosis infection. Most activities exerted by TNF were originally attributed to the soluble form of TNF (solTNF). The generation of uncleavable memTNF by mutations in the region cleaved by the TACE (TNF-a converting enzyme) and subsequent studies on cells, transgenic mice and later in memTNF knockin (KI) mice without soluble TNF have shown the importance of activities mediated by the transmembrane form of TNF (memTNF). Several memTNF activities in immune responses to intracellular bacterial infection have been shown to be different from those exerted by soluble TNF. Evidence is based largely on studies in transgenic mice expressing memTNF, but precise cellular mechanisms are not well defined and the importance of TNF receptor regulation is unknown. In addition, memTNF activities are defined for a particular modification of the extracellular domain of TNF but a direct comparison of different mutant memTNF molecules has not been done in vivo . To understand the activities of memTNF we compared two mouse strains lacking soluble TNF but possessing functional and normally regulated membrane-bound TNF knockin (memTNF KI) for their capacity to generate cell-mediated immune responses and resistance to M. bovis BCG infection, and to regulate TNF receptors. M. bovis BCG infection resulted in similar bacterial loads in one strain of memTNF KI (memTNF Δ1–9, K 11 E ) and in wild-type mice, in contrast, the other strain of memTNF KI mice (memTNF Δ1–12 ) showed higher sensitivity to infection with high mortality (75%), greater bacterial load and massive lung pathology. The pattern of cytokines/chemokines, inflammatory cells, pulmonary NF-kB phosphorylation, antigen-dependent IFN-g response, and splenic iNOS was impaired in M. bovis BCG-infected memTNF Δ1–12 KI mice. Macrophages expressing TNFR2 were reduced but soluble TNFRs were higher in memTNF Δ1–12 KI mice during the infection. In vitro , M. bovis BCG-induced NF-kB activation and cytokines were also decreased in memTNF Δ1–12 KI bone marrow-derived macrophages. Our data show that two memTNF molecules exerted very different activities upon M. bovis BCG infection resulting in protection or not to bacterial infection. These results suggest a regulatory mechanism of memTNF and TNF receptors being critical in the outcome of the infection and highlight the role of cell-bound and soluble TNFR2 in memTNF-mediated anti-microbial mechanisms.

A novel role for iNOS in formation of cGMP-dependent protein complex regulating TNF-receptor shedding and hepatocellular protection during endotoxemia

TNFα is produced in significant quantities during endotoxemia and can induce liver cell apoptosis. Type 1 TNF-receptor (TNFR1) can be cleaved from the cell surface by TACE/ADAM17 allowing binding to excess TNF, and reducing liver damage, although the mechanism of TACE activation is unknown. Inducible nitric oxide synthase (iNOS) is also hepatoprotective during endotoxemia. The aim of this study was to identify whether iNOS-dependent signaling regulates TACE activation and TNFR1-shedding for hepatocellular protection. Primary mouse hepatocytes were isolated from C57BL/6 mice and treated for 12 h with LPS (100 ng/mL) and NO donor SNAP (80 μM). TACE activity and soluble TNFR1(sTNFR1) levels in culture media significantly increased after just 30 min with LPS/SNAP compared to controls (p = 0.002). Both iNOS inhibitor (1400 W–2.5 μM) and soluble guanylate cyclase inhibitor (ODQ-20 μM) blocked TACE activity and TNFR1-shedding. Conversely, hepatocytes treated with cell-permeable cGMP-analog (8-Br-cGMP-800 μM) had significantly increased TACE activity and sTNFR1 after 30 min compared with untreated controls (p = 0.015). This increase in sTNFR1 was significantly reduced after addition of either ODQ or Protein Kinase G-inhibitor (PKG-inhibitor-20 μM). Coimmunoprecipitation experiments revealed iNOS, activated TACE, TNFR1 and PKG are mutually associated at the hepatocyte membrane after stimulation with LPS, and this association is required for TACE activation. Furthermore, treatment of protein extracts with ODQ, PKG inhibitor or TAPI-1 (TACE inhibitor) prevented formation of the complex. Taken together our findings suggest that iNOS-derived cGMP induces the shedding of cell surface TNFR1 through the formation of a multi-protein membrane-associated complex. These data shed light on the mechanism of hepatoprotection by iNOS during infection and inflammation.

Membrane-Bound TNF Induces Protective Immune Responses to M. bovis BCG Infection: Regulation of memTNF and TNF Receptors Comparing Two memTNF Molecules

BackgroundSeveral activities of the transmembrane form of TNF (memTNF) in immune responses to intracellular bacterial infection have been shown to be different from those exerted by soluble TNF. Evidence is based largely on studies in transgenic mice expressing memTNF, but precise cellular mechanisms are not well defined and the importance of TNF receptor regulation is unknown. In addition, memTNF activities are defined for a particular modification of the extracellular domain of TNF but a direct comparison of different mutant memTNF molecules has not been done in vivo.MethodologyTo understand the activities of memTNF we compared two commonly used mouse strains lacking soluble TNF but possessing functional and normally regulated membrane-bound TNF knockin (memTNF KI) for their capacity to generate cell-mediated immune responses and resistance to M. bovis BCG infection, and to regulate TNF receptors.Principal Findings M. bovis BCG infection resulted in similar bacterial loads in one strain of memTNF KI (memTNFΔ1–9,K11E) and in wild-type mice, in contrast, the other strain of memTNF KI mice (memTNFΔ1–12) showed higher sensitivity to infection with high mortality (75%), greater bacterial load and massive lung pathology. The pattern of cytokines/chemokines, inflammatory cells, pulmonary NF-κB phosphorylation, antigen-dependent IFN-γ response, and splenic iNOS was impaired in M. bovis BCG-infected memTNFΔ1–12 KI mice. Macrophages expressing TNFR2 were reduced but soluble TNFRs were higher in memTNFΔ1–12 KI mice during the infection. In vitro, M. bovis BCG-induced NF-κB activation and cytokines were also decreased in memTNFΔ1–12 KI bone marrow-derived macrophages.ConclusionOur data show that two memTNF molecules exerted very different activities upon M. bovis BCG infection resulting in protection or not to bacterial infection. These results suggest a regulatory mechanism of memTNF and TNF receptors being critical in the outcome of the infection and highlight the role of cell-bound and soluble TNFR2 in memTNF-mediated anti-microbial mechanisms.

Stage-related changes of peritoneal soluble TNFα and TNFR1 and TNFR2 in cells recovered from peritoneal fluid of women with endometriosis

B. Gharesi-Fard1,2,∗, F. Ghaderi-shabankareh1, L. Jafarzadeh1, J. Zolghadri3,2, E. Kamali-Sarvestani1,4 1 Shiraz University of Medical Sciences, Immunology Department, Shiraz, Iran, Islamic Republic of 2 Infertility Research Center, Shiraz, Iran, Islamic Republic of 3 Shiraz University ofMedical Sciences, Department of Obstetrics and Gynecology, Shiraz, Iran, Islamic Republic of 4 Autoimmune Diseases Research Center, Shiraz, Iran, Islamic Republic of

Abstract 397: Soluble Tumor Necrosis Factor Receptor shed by breast tumor cells modulates macrophage migration

Abstract Tumor necrosis factor alpha (TNFα) present within the tumor microenvironment, especially in breast tumor with a strong inflammatory component, promotes tumor death. TNFα also stimulates the migration of immune cells including macrophages to the tumor site. Breast cancer cells express both TNFR1 and TNFR2, the cell surface receptors for TNFα. The breast tumor microenvironment also contains soluble forms of TNFR1 and TNFR2. We investigated the origins and effects of soluble TNFR1 and TNFR2 on monocyte/macrophage cell migration. Our results indicate that both sTNFR1 and sTNFR2 were shedded by tumor cells but also by stroma cells including adipocytes. In addition, the amounts of sTNFR2 shedded by adipocytes and 4T1 mammary tumor cells were significantly higher than the amounts of sTNFR2 shedded by normal murine epithelial NMuMG cells (p&amp;lt;0.05). The migration of M2 subtype macrophages but not of M1 subtype macrophages derived from either murine bone derived monocytes, J774 or Raw cells toward 4T1 mammary tumor cells’ conditioned media was significantly increased following treatment of 4T1 cells with the sheddase inhibitor TAPI-0 (p&amp;lt;0.05). Indeed, TAPI-0 treatment decreased sTNFR2 concentrations in 4T1-conditioned media (p&amp;lt;0.05). These observations suggest a role for sTNFR1 and sTNFR2 in the dynamic interplays between immune and tumor cells during breast progression. (This works is supported by grants from the Department of Defense Era of Hope research program (BC044778) and the National Science Foundation EFRI program (CBE0736007) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 397. doi:1538-7445.AM2012-397

Abstract C10: Tumor necrosis factor is necessary but not sufficient for castration-induced prostate regression

Abstract TNF, a pro-inflammatory and immune-regulatory cytokine, is a potent apoptotic stimulus in vitro. There are few examples of a physiologic role for TNF induced apoptosis in vivo, but here we describe a novel role for TNF in prostate epithelial cell apoptosis following androgen withdrawal. We demonstrate, using high resolution serial MRI (magnetic resonance imaging) to measure mouse prostate volume changes over time, that castration induced prostate regression is significantly reduced in mice null for either the Tnf or Tnfr1 genes, but not mice deficient for TRAIL or Fas signaling. Wild type mice treated with soluble TNFR2 (to bind TNF and block signaling) prior to castration showed a similar reduction. Additionally, consistent with a paracrine role for TNF in prostate regression, membrane-bound TNF protein and stromal cell specific TNF mRNA levels increase in rat prostate following castration. Injection of soluble TNF restores normal levels of prostate regression of Tnf −/− mice. Together, these data indicate that uniquely among these extrinsic death signals, TNF is required for castration induced prostate regression. However, wild type mice receiving soluble TNF in the absence of castration do not exhibit prostate regression, indicating that TNF alone is not sufficient, but acts in the context of additional castration induced signals. A preliminary investigation using ALK5 inhibitors prior to castration suggests that TGFβ signaling may also be important. Understanding the physiologic role for TNF, and the interaction with other signaling pathways active in prostate regression following androgen withdrawal may lead to novel therapies for prostate cancer. Citation Format: Jennifer S. Davis, Kent L. Nastiuk. Tumor necrosis factor is necessary but not sufficient for castration-induced prostate regression [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C10.

Increased tumor necrosis factor receptor 1 expression in human colorectal adenomas

To determine the expression statuses of tumor necrosis factor (TNF)-α, its receptors (TNF-R) and downstream effector molecules in human colorectal adenomas. We measured the serum concentrations of TNF-α and its receptors in 62 colorectal adenoma patients and 34 healthy controls. The protein expression of TNF-α, TNF-R1, TNF-R2 and downstream signals of the TNF receptors, such as c-Jun N-terminal kinase (JNK), nuclear factor-κ B and caspase-3, were also investigated in human colorectal adenomas and in normal colorectal mucosal tissues by immunohistochemistry. Immunofluorescence confocal microscopy was used to investigate the consistency of expression of TNF-R1 and phospho-JNK (p-JNK). The serum levels of soluble TNF-R1 (sTNF-R1) in adenoma patients were significantly higher than in the control group (3.67 ± 0.86 ng/mL vs 1.57 ± 0.72 ng/mL, P < 0.001). Receiver operating characteristic analysis revealed the high diagnostic sensitivity of TNF-R1 measurements (AUC was 0.928) for the diagnosis of adenoma, and the best cut-off level of TNF-R1 was 2.08 ng/mL, with a sensitivity of 93.4% and a specificity of 82.4%. There were no significant differences in the serum levels of TNF-α or sTNF-R2 between the two groups. Immunohistochemistry showed high levels of TNF-R1 and p-JNK expression in the epithelial cells of adenomas. Furthermore, a high incidence of co-localization of TNF-R1 and p-JNK was identified in adenoma tissue. TNF-R1 may be a promising biomarker of colorectal adenoma, and it may also play an important role in the very early stages of colorectal carcinogenesis.